Severe bleeding for organ resection, trauma, or large dermal wounds is sometimes difficult to control. Recently fibrin-based patches have been tested to seal grade IV/V wounds using cellulose or gelatin supports. These technologies however cannot be applied to certain types of procedures such as interventional radiology, orthopedic or laparoscopic surgery. In addition the “support” used by these products remains in the body until biodegraded causing severe inflammatory reaction to foreign body.
Current Solutions and Limitations.
In general, the synthetic adhesives are used for the tight sealing of vessels or of lungs and for “gluing” the edges of skin incisions. These glues are eliminated, in general after the scaring of the wound, by biodegradation, absorption or by simple detachment in the form of scabs. Various technologies have been developed for the formulation of tissue adhesives. Some of them are of synthetic origin, such as the glues based on cyanoacrylates (2-butyl cyanoacrylate, 2-octyl cyanoacrylate), or synthetic polymers, and others contain biological materials such as collagen or fibrin which in addition have hemostatic properties.
As a result of their hemostatic and adhesive properties, sealants, and particularly fibrin sealants have been extensively used in most surgical specialties for over two decades to reduce blood loss and post-operative bleeding because of the ability to adhere to human tissue as they polymerize (1, 2, 3). These compounds are used to seal or reinforce the sealing of wounds that have been sutured or stapled; they can also be used with pressure over an injured area. Fibrin sealants are biological adhesives that mimic the final step of the coagulation cascade. (4) The main components of the sealant are fibrinogen, plasma proteins and factor XIII on the one hand and thrombin, and calcium chloride on the other. The components are often extracted from human plasma or produced by recombinant techniques. Mixing fibrinogen and thrombin creates a polymer barrier (fibrin) that simulates the last stages of the natural coagulation cascade to form a structured fibrin clot similar to a physiological clot.
There are several commercial products available (Floseal, Gelfoam, Evicel, Bioglue, surgicel, tachoseal, etc) (3, 5). However, these products have significant limitations, which have prevented their widespread use in cases of severe bleeding in surgery and in emergency medicine, orthopedic and interventional radiology and laparoscopic surgery. All existing haemostatic agents for intracavitary bleeding are designed to be used as adjuncts in light to moderate bleeding and require hard compression. One of the major limitations encountered in the development and/or use of tissue adhesive and sealant compositions for minimally compressible hemorrhage is their inability to form a sufficiently strong bond to tissues when there is profuse bleeding and to produce a stable clot within 10 minutes of application. Therefore, tissue adhesives and sealants have to be employed in combination with compression methods, sutures and/or staples, and adhesive patches so as to reduce the tissue-bonding strength required for acceptable performance. However, there are many situations where the use of strong compression, sutures and/or staples is undesirable, inappropriate or impossible, (e.g. in bone, interventional radiology).
The Present Alternative Approach:
In order to form a physical barrier that resists the flow of blood, the adhesive matrix must form in a matter of seconds a strong fibrin interface, bond with tissues in the midst of flowing blood and remain at the lacerated site to form a clot. The ability to adhere to human tissue of each of the product presentations in a form of a square; a round patch a sphere, a cylinder, or a cone is related to the composition and method of production of fibrin and its interaction (combination) with thrombin to stimulate the coagulatory cascade. The essential aspect of the technology is the ability to bypass the cleavage process of fibrinogen to produce a fibrin monomer and its subsequent polymerized, lyophilized and capable to absorb blood to form a fibrin clot. The agent starts the Clot formation process from an already stabilized I fibrin polymer that absorb the blood into a lyophilized crosslinked polymer containing the necessary components to stimulate the coagulatory cascade (thrombin) and form a physical barrier that turns into a functional fibrin clot within two minutes of application. (6)
In our approach these results are obtained through a) the application of a polymeric cross-linked lyophilized fibrin network that absorbs the blood forming a very sticky gel-like matrix, which attaches to lacerated tissue; and b) the incorporation of a thrombin layer that contributes to the rapid formation of a strong fibrin clot stabilized by calcium independent transglutaminase enzyme incorporated in the product, and by Factor XIII from the blood.
The lyophilization process in subsequent layers over a biodegradable removable support facilitates its application, and allows for long-term storage, transportation and readiness.
In addition, no fibrin-based products have been developed to address the need arresting hemorrhage from deep cuts or large bed cutaneous wounds, which cause such severe bleeding that often require stitches or sutures.
Composition.
All the forms of the present technology—square, sphere, cylinder or cone incorporate fibrin monomer in acid solution polymerized by a change of pH, which is neutralized by a buffer solution in the presence of activated transglutaminase enzyme (calcium independent) and Factor XIII (calcium dependent) (Calcium dependent and Calcium independent), and calcium chloride. Once fibrin polymer is formed and cross-linked, a second layer of thrombin is incorporated and subsequently lyophilized. (FIG. 1)
The monomer can be mixed with a volume of about 1% to about 5% of glycerol to achieve a specific viscoelastic profile that is adapted to the type of application. The absorption of blood by the cake turns the lyophilized fibrin into a gel, which forms the fibrin clot at sites of injury (7).
Under coagulant conditions, calcium independent transglutaminase and activated Factor XIII from the blood contribute to this process by stabilizing the fibrin clot through covalent bonds.
Key Attributes. Polymerization/Adhesion.
The fibrin gel that seals the wound is formed as a result of the absorption of blood by the lyophilized bilayer material, which maintains covalent bonds while changing from solid to gel state. The clot is mechanically stable, well integrated into the wound and more resistant to lysis by plasmin compared with a non-cross-linked clot [8] or other fibrin sealants. The inclusion of calcium independent transglutaminase facilitates the transglutaminase-mediated cross-linking of the aC-domains polymers in fibrin promoting integrin clustering and thereby increasing cell adhesion and spreading, which stimulates fibrin to bind avb3-, avb5- and a5b1-integrins on endothelial cells [9]. The oligomerization also promotes integrin-dependent cell signaling via focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), which results in an increased cell adhesion and cell migration [10]. The presence of calcium ions enhances the progression from the inflammatory response to the coagulation cascade (first stage) and activates Factor XIII.
The adhesion characteristics to vital human tissue and the kinetics of polymerization of the proposed agent have been tested in vitro and in vivo. The data obtained provide ample evidence of the ability of the various presentation of ClotBlock to stop bleeding and achieve hemostasis with minimal compression in induced intraperitoneal wounds in solid organs or soft tissue. And to stop intramedulary bone bleeding in knee and hip replacement in the swine models.
Lyophilized Fibrin II is obtained from fibrin II monomer polymerization: U.S. patent application Ser. No. 12/487,057 (allowed), which describes a method of preparing a fibrin monomer. The ClotBlock sealant composition uses a lyophilized fibrin polymer obtained from neutralization of fibrin monomer. The composition of parts and method of production of the fibrin II described in this patent application as well as the process of neutralization and crosslinking of the polymer are critical to the performance of the proposed technology which depends on the characteristics of fibrin itself (thickness of the fibers, the number of branch points, the porosity, and the permeability and other polymerization characteristics define clotting factors. The Clot produced by ClotBlock creates opaque matrices of thick fibers, and therefore tube formation proceeds at a faster rate than in transparent matrices. The concentration of thrombin to produce a fibrin monomer and thus the release-rate of FPA also has an important impact on the polymerization process. The described concentrations, dilutions and pH established for ClotBlock produce an optimal fibrin structure at an accelerated rate.
ClotBlock Presentations:
The fibrin polymer can be produced and lyophilized in various sizes, thickness and forms in order to adapt to the type of application (FIG. 2A, 2B, 2C). It can be configured in small spheres or cylinders ¼ inch diameter to be introduced through a laparoscopic port or a vessel in cases on interventional radiology; it can also be molded in round or square flat solid blocks of various sizes in ¼; ½ a 1″ thickness for use in spleen laceration, or organ resection, or placed over an adhesive bandage to cover deep skin cuts. The lyophilized form can also be soaked in water and used as a sealing paste or gel.